Katada T and Kinoshita T (2003), XMam1, the Xenopus homologue of mastermind, is ...

XB-ART-4492

Int J Dev Biol 2003 Sep 01;476:397-404.

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XMam1, the Xenopus homologue of mastermind, is essential to primary neurogenesis in Xenopus laevis embryos.

Katada T , Kinoshita T .

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Notch signaling is involved in cell fate determination and is evolutionally highly conserved in vertebrates and invertebrates. Mastermind is a nuclear protein which participates in Notch signaling and is involved in direct transactivation of target genes. Here we analyzed the expression and the function of Xenopus mastermind1 (XMam1) in the process of primary neurogenesis. XMam1 is 3,425 bp and encodes 1,139 amino acids. Overall, Mastermind proteins consist of a basic domain, two acidic domains and a glutamine-rich domain, which are highly conserved among species. The ubiquitous expression of XMam1 was observed in both maternal and zygotic stages. Whole-mount in situ hybridization showed that XMam1 mRNA was present in the ectoderm by the gastrula stage and localized at the anterior neural region in the neurula stage. Thereafter, XMam1 expression was restricted to the eye and otic vesicle in the tailbud-stage embryo. XMaml overexpression caused the repression of primary neural formation. The truncated form of XMam1 (lacking the C-terminus of XMam1; XMam1deltaC) led to excess formation of primary neurons. Furthermore, XMam1deltaC strongly repressed XESR-1 transactivation. These results show that XMaml is involved in primary neurogenesis by way of Notch signaling and is an essential component for transactivation of XESR-1 in Xenopus laevis embryos.

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Species referenced: Xenopus laevis
Genes referenced: hes5.1 maml1 notch1 rbpj smpx tbx2 tubb2b

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	Fig. 4. The spatial expression of XMam1 analyzed by whole-mount in situ hybridization. The staining in brown shows XMam1 expression. (A) Blastula (st. 7). Lateral view. XMam1 is expressed in the animal hemisphere. (B) Animal view of (A). (C) Mid-gastrula (st. 11). Lateral view. The expression of XMam1 was more intense in the animal half than in the vegetal half. Arrow indicates blastopore. (D) Animal view of (C). (E) Mid-neurula (st. 15). Dorsal view. XMam1 is expressed in the anterior side of embryos. (F) Anterior view of (E). XMam1 mRNA was observed at the anterior side of the neural plate. (G) Late neurula (st. 20). Dorsal view. As in mid-neurula, transcripts of XMam1 were detected at the anterior side. A weak XMam1 mRNA signal was observed in the neural tube. White line indicates the plane of section of panel (M). (H) Anterior view of (G). In comparison to expression in st. 15 embryos, the XMam1-expressing region was somewhat narrower. (I) st. 25 embryo. Lateral view. Strong expression of XMam1 was observed in the eye, in addition to the head regions. (J) Dorsal view of (I). (K) Tailbud-stage embryo (st. 35). Lateral view. XMam1 expression was observed in the entire region of head. The intensity of XMam1 transcripts observed at st. 25 was reduced at this stage. (L) Magnification of the head in (K). XMam1 was strongly expressed in the otic vesicle (arrowhead). (M) Transverse section of embryo shown in (G). XMam1 was expressed in the neural tube. nt, neural tube; n, notochord; so, somite.
	maml1 (mastermind-like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 7, lateral view, animal up.
	maml1 (mastermind-like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior left.
	maml1 (mastermind-like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.
	maml1 (mastermind-like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, cross section through middle of embryo.
	maml1 (mastermind-like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	maml1 (mastermind-like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anterior left, dorsal up.
	Fig. 5. The effect of XMam1 on N-tubulin expression. N-tubulin expression was detected by whole-mount in situ hybridization (stained in brown). Samples were injected into the blastomere of two-cell stage embryos and N-tubulin expression analyzed at st. 14. β-galactosidase was used as tracer (the injected side appeared blue as indicated by arrows). (A) Control embryo injected with β-galactosidase. (B) X-Delta-1-injected embryo. The suppression of primary neurons was observed in the injected side. (C) XMam1-injected embryo. Primary neurogenesis was repressed in the injected side. (D) XMam1∆C-injected embryo. The overproduction of primary neurons was observed in the injected side.
	Fig. 6. The effect of XMam1 on XESR-1 expression. XESR-1 expression was detected by whole-mount in situ hybridization (stained in brown). Samples were injected at one blastomere of two-cell stage embryos and XESR-1 expression analyzed at st. 14. β-galactosidase was used as tracer (injected side was appeared in blue as indicated by arrows). (A) Control embryo injected with β-galactosidase. (B) X-Delta-1-injected embryo. The ectopic expression was observed in injected side. (C) XMam1-injected embryo. Overexpression of XMam1 caused no change of XESR-1 expression pattern. (D) XMam1∆C-injected embryo. The remarkable reduction of XESR-1 expression was observed in injected side.
	Fig. 7. Repression of XESR-1 expression in embryos by XMam1∆C is rescued by XMam1. XESR-1 expression was detected by whole-mount in situ hybridization (stained in brown). Samples were injected into the blastomere of two-cell stage em- bryos and XESR-1 expression was analyzed at st. 14. β-galactosidase was used as a tracer (the injected Discussion In this report, we identified Xenopus homologue of mastermind, XMam1, which is involved in transactivation of target genes by Notch signaling and analyzed the structure, expression profile and the role of XMam1 in the process of primary neurogenesis. In vertebrates, the function of Mastermind is not understood yet, especially in develop- mental process. Therefore, our findings are valuable on analyzing the function of Mastermind in primary neurogenesis of vertebrate development. Identification of XMam1, the Xenopus homologue of master- mind1 The EST clone (AW765543) we identified included one open reading frame and encoded 1139 amino acids. The comparison of this clone with three kinds of human Mastermind in amino acid sequence revealed that this clone had the highest homology to human Mastermind1 (hMam1). Furthermore, The homology in Mas- termind-specific domain, basic domain and two acidic domains between these two, was also highest. Therefore, this clone AW765543 was designated as Xenopus homologue of mastermind, XMam1. Amino acids in the other domain except Mastermind-specific domain were not identical but still similar. This evidence will also support that this clone is Xenopus homologue of mastermind1. In human Mastermind, it is reported that there exists three kinds of Mastermind, but the homology of each molecule is not so high in amino acid level (Lin et al., 2002; Wu et al., 2002). In Mastermind- specific domain, hMam1 and hMam2 shares with high homology relatively, but hMam3 has little homology to hMam1 and hMam2. These suggest that XMam1 form a new gene family. Different types of Mastermind may have different regulatory mechanism by different types of Notch signals in various developmental stages and places. The role of XMam1 in transactivation of the Notch target gene In our experiments, we found that XMam1∆C, XMam1 lacking of two acidic domains, intensely repressed transactivation of XESR-1. Additionally, we found that XESR-1 repression by XMam1∆C was rescued by XMam1. Therefore, it is thought that this effect is XMam1- specific. These results show that XMam1 is essential molecule on transactivation of XESR-1. Consistent with this result, Fryer reported that Mastermind is essential to in vitro transactivation in complex of Notch-ICD (NICD) and CBF1 on chromatin (Fryer et al., 2002). These data strongly support our experimental result and Mastermind is essential to transactivation of target genes by Notch signaling in vivo. In addition to this, the idea that two acidic domains in XMam1 contribute transactivation of Notch target gene coincides with the experimental data reported previously (Wu et al., 2000; Kitagawa et al., 2001; Fryer et al., 2002; Lin et al., 2002; Wu et al., 2002). Therefore, It is thought that Mastermind recognizes and binds to NICD-CSL complex through basic domain, and regulates transcription of target genes through acidic domain. As additional interest, in this experiment, XMam1 overexpression resulted in the repression of primary neurogenesis although XMam1 did not enhance XESR-1 transcription. We discuss about this in next section. The regulatory mechanism of primary neurogenesis It is reported that Notch signaling is deeply involved in primary neurogenesis and overexpression of components of Notch signal- side appeared in blue as indicated by arrows). (A) Control embryo injected with β-galactosidase. (B) Embryo injected with 1ng of XMam1∆C. Down- regulation of XESR-1 expression was observed in the injected side.
	Fig. 7. Repression of XESR-1 expression in embryos by XMam1∆C is rescued by XMam1. XESR-1 expression was detected by whole-mount in situ hybridization (stained in brown). Samples were injected into the blastomere of two-cell stage em- bryos and XESR-1 expression was analyzed at st. 14. β-galactosidase was used as a tracer (the injected Discussion In this report, we identified Xenopus homologue of mastermind, XMam1, which is involved in transactivation of target genes by Notch signaling and analyzed the structure, expression profile and the role of XMam1 in the process of primary neurogenesis. In vertebrates, the function of Mastermind is not understood yet, especially in develop- mental process. Therefore, our findings are valuable on analyzing the function of Mastermind in primary neurogenesis of vertebrate development. Identification of XMam1, the Xenopus homologue of master- mind1 The EST clone (AW765543) we identified included one open reading frame and encoded 1139 amino acids. The comparison of this clone with three kinds of human Mastermind in amino acid sequence revealed that this clone had the highest homology to human Mastermind1 (hMam1). Furthermore, The homology in Mas- termind-specific domain, basic domain and two acidic domains between these two, was also highest. Therefore, this clone AW765543 was designated as Xenopus homologue of mastermind, XMam1. Amino acids in the other domain except Mastermind-specific domain were not identical but still similar. This evidence will also support that this clone is Xenopus homologue of mastermind1. In human Mastermind, it is reported that there exists three kinds of Mastermind, but the homology of each molecule is not so high in amino acid level (Lin et al., 2002; Wu et al., 2002). In Mastermind- specific domain, hMam1 and hMam2 shares with high homology relatively, but hMam3 has little homology to hMam1 and hMam2. These suggest that XMam1 form a new gene family. Different types of Mastermind may have different regulatory mechanism by different types of Notch signals in various developmental stages and places. The role of XMam1 in transactivation of the Notch target gene In our experiments, we found that XMam1∆C, XMam1 lacking of two acidic domains, intensely repressed transactivation of XESR-1. Additionally, we found that XESR-1 repression by XMam1∆C was rescued by XMam1. Therefore, it is thought that this effect is XMam1- specific. These results show that XMam1 is essential molecule on transactivation of XESR-1. Consistent with this result, Fryer reported that Mastermind is essential to in vitro transactivation in complex of Notch-ICD (NICD) and CBF1 on chromatin (Fryer et al., 2002). These data strongly support our experimental result and Mastermind is essential to transactivation of target genes by Notch signaling in vivo. In addition to this, the idea that two acidic domains in XMam1 contribute transactivation of Notch target gene coincides with the experimental data reported previously (Wu et al., 2000; Kitagawa et al., 2001; Fryer et al., 2002; Lin et al., 2002; Wu et al., 2002). Therefore, It is thought that Mastermind recognizes and binds to NICD-CSL complex through basic domain, and regulates transcription of target genes through acidic domain. As additional interest, in this experiment, XMam1 overexpression resulted in the repression of primary neurogenesis although XMam1 did not enhance XESR-1 transcription. We discuss about this in next section. The regulatory mechanism of primary neurogenesis It is reported that Notch signaling is deeply involved in primary neurogenesis and overexpression of components of Notch signal- side appeared in blue as indicated by arrows). (C) Embryo co-injected with 1ng of XMam1∆C and 3 ng of XMam1. XESR-1 expression was restored.